Non-Invasive Fetal Genetic Screening by Digital Analysis

ABSTRACT

The present methods are exemplified by a process in which maternal blood containing fetal DNA is diluted to a nominal value of approximately 0.5 genome equivalent of DNA per reaction sample. Digital PCR is then be used to detect aneuploidy, such as the trisomy that causes Down Syndrome. Since aneuploidies do not present a mutational change in sequence, and are merely a change in the number of chromosomes, it has not been possible to detect them in a fetus without resorting to invasive techniques such as amniocentesis or chorionic villi sampling. Digital amplification allows the detection of aneuploidy using massively parallel amplification and detection methods, examining, e.g., 10,000 genome equivalents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional PatentApplication No. 60/764,420 filed on Feb. 2, 2006, and U.S. Utilitypatent application Ser. No. 11/701,686, filed Feb. 2, 2007, which arehereby incorporated by reference in their entirety.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with U.S. Government support under contract0535870 awarded by the National Science Foundation. The U.S. Governmenthas certain rights in this invention.

REFERENCE TO SEQUENCE LISTING

Applicants assert that the paper copy of the Sequence Listing isidentical to the Sequence Listing in computer readable form found on theaccompanying computer file. Applicants incorporate the contents of thesequence listing by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of fetal genetic screeningand to the field of quantitative nucleic acid analysis.

2. Related Art

It is now recognized that fetal DNA sheds from the placenta and mixeswith the mother's blood at fairly high levels—between 3% and 6% of DNAin the mother's blood is from the fetus. This observation has been usedin conjunction with PCR assays for a variety of fetal geneticscreens—gender, Rh, and thalassemia. However, the technique remainslimited for two primary reasons: first, the PCR assays trade offsensitivity for specificity, making it difficult to identify particularmutations, and second, the most common genetic disorder, Down Syndrome,is a chromosomal trisomy and therefore cannot be detected byconventional PCR in a mixed sample.

It has now been found that these problems can be solved by quantitativeexamination of large numbers of chromosome samples through the use ofhighly scalable techniques. This approach is termed here “digitalanalysis,” and involves the separation of the extracted genomic materialinto discrete units so that the detection of a target sequence (e.g.,chromosome 21) may be simply quantified as binary (0, 1) or simplemultiples, 2, 3, etc. The primary example of a technique that can beused to yield such “digital” results is “digital PCR,” which allowsefficient amplification from single molecules, followed by subsequentquantitative analysis. Digital PCR, as the term is used here, refers toa quantitative, limited dilution of a nucleic acid sample, such as intomultiwell plates, then the amplification of a nucleic acid molecule in awell, which due to the dilution, should be either 0 or 1 molecule.Digital PCR using multiwell plates has been used previously to detectrare mutations by either serial analysis of single molecule (i.e.,clonal) amplicons (Vogelstein B, Kinzler K W. Proc Natl Acad Sci USA.1999 Aug. 3; 96 (16): 9236-41) or by enhancing the sensitivity ofdifferential amplification (http://www.fluidigm.com/didIFC.htm).Described below is an invention whereby digital PCR can be applied tononinvasive fetal diagnostics in order to detect fetal mutations withspecificity and sensitivity beyond what is possible with conventionalPCR analysis.

Furthermore, as also described in connection with the inventiondescribed below, digital PCR can be used to detect aneuploidy, such asthe trisomy that causes Down Syndrome. Since aneuploidies do not presenta mutational change in sequence, and are merely a change in the numberof chromosomes, it has not been possible to detect them in a fetuswithout resorting to invasive techniques such as amniocentesis orchorionic villi sampling (Science 309, 2 Sep. 2005 pp. 1476-8).

Another form of digital PCR has been described as emulsion PCR, whichhas been used to prepare small beads with clonally amplified DNA—inessence, each bead contains one amplicon of digital PCR. (Dressman etal, Proc Natl Acad Sci USA. 100, 8817 (Jul. 22, 2003)).

Another form of Digital PCR can be carried out using microfluidics. Inthis embodiment, described below, DNA is diluted and separated intosmall, discrete samples for forming reaction samples by a series ofchannels and valves.

An example of a suitable method for single molecule analysis that may beadapted to the present methods is given in Braslavsky et al., “Sequenceinformation can be obtained from single DNA molecules, Proc. Nat. Acad.Sci. 100(7): 3960-3964 (2003), which uses sequential incorporation oflabeled nucleotides onto an immobilized single stranded DNA template andmonitoring by fluorescent microscopy.

Another aspect of the relevant art involves sample preparation in orderto carry out the present processes. That is, the fetal DNA may beenriched relative to maternal DNA. Chan, et al., “Size Distribution ofMaternal and Fetal DNA in Maternal Plasma,” Clin. Chem. 50(1): 88-92(2004) reports that plasma DNA molecules are mainly short DNA fragments.The DNA fragments in the plasma of pregnant women are significantlylonger than DNA fragments from non-pregnant women, and longer than fetalDNA.

Related Publications and Patents

Vogelstein et al., “Digital Amplification,” U.S. Pat. No. 6,440,705,issued Aug. 27, 2002, discloses the identification of pre-definedmutations expected to be present in a minor fraction of a cellpopulation.

Lo, “Fetal DNA in Maternal Plasma: Biology and Diagnostic Applications,”Clin. Chem. 46:1903-1906 (2000) discloses the demonstration of fetal DNAin maternal plasma. The authors found a mean fractional level of 3.4%fetal DNA in maternal DNA in plasma during early pregnancy. The authorsreport detection of the RhD gene and microsatellite polymorphisms in theplasma of pregnant women.

Li et al., “Detection of Paternally Inherited Fetal Point Mutations forβ-Thalassemia Using Size Fractionated Cell-Free DNA in Maternal Plasma,”J. Amer. Med. Assoc. 293:843-849 (Feb. 16, 2005) discloses that theanalysis of cell-free fetal DNA in maternal plasma has proven to beremarkably reliable for the assessment of fetal loci absent from thematernal genome, such as Y-chromosome specific sequences or the RhD genein pregnant women who are Rh-negative. The authors report on theextraction and size fractionation of maternal plasma DNA using agarosegel electrophoresis. Then, peptide-nucleic acids (PNA) were used to bindspecifically to a maternal allele to suppress PCR amplification of theof the wild type maternal allele, thereby enriching for the presence ofpaternally inherited mutant sequences. Four distinct point mutations inthe β-globin gene were examined. It was found that the PNA step wasnecessary for the detection of mutant alleles using allele specific PCR.

Lo et al., “Quantitative Analysis of Fetal DNA in Maternal Plasma andSerum: Implications for Noninvasive Prenatal Diagnosis,” Am. J. Hum.Genet. 62:768-775 (1998) discloses a real-time quantitative PCR assay tomeasure the concentration of fetal DNA in maternal plasma and serum. Theauthors found a mean of 25.4 genome equivalents/ml of fetal DNA in earlypregnancy. This corresponds to about 3.4% of total DNA in earlypregnancy.

Chan et al., “Size Distribution of Maternal and Fetal DNA in MaternalPlasma,” Clin. Chem. 50:89-92 (January 2004) investigated the sizedistribution of plasma DNA in non-pregnant women and pregnant women,using a panel of quantitative PCR assays with different amplicon sizestargeting the leptin gene. They found that the DNA fragments in theplasma of pregnant women are significantly longer than those in theplasma of non-pregnant women, and the maternal-derived DNA molecules arelonger than the fetal-derived ones.

Tufan et al., “Analysis of Cell-Free Fetal DNA from Maternal Plasma andSerum Using a Conventional Multiplex PCR: Factors Influencing Success,”Turk. J. Med. Sci. 35: 85-92 (2005) compared the success rates of twodifferent DNA extraction techniques, the heat based direct method andthe QIAMP DNA blood mini kit method. The crucial role of PCRoptimization was also reported. The authors used the DYS14 marker forthe Y chromosome and the GAPH gene for a control. The QIAMP mini kit wasfound to give the best results in sex determination analysis usingmultiplex PCR and ethidium bromide staining on gels.

Hromadnikova et al., “Quantitative analysis of DNA levels in maternalplasma in normal and Down Syndrome pregnancies,” BMC Pregnancy andChildbirth 2(4): 1-5 (2002), investigated total DNA levels in maternalplasma and found no difference in fetal DNA levels between the patientscarrying Down Syndrome fetuses and the controls. Real time quantitativePCR analysis was performed using primers to the β-globin gene and theSRY locus.

Grundevikk and Rosen, “Molecular Diagnosis of Aneuploidies,” publishedon line at http://www.molbiotech.chalmers.se/research/mk/mbtk/Molecular%20diagnostics%20of%20aneuploidies%20-%20rapport.pdf, suggests that non-invasive methodsfor detection of aneuploidies (such as Down Syndrome, Edwards Syndromeor extra sex chromosomes) may be carried out on fetal nucleated cellsisolated from maternal blood. In their review, the authors also describequantitative fluorescence polymerase chain reaction (QF-PCR), based onamplification of short tandem repeats specific for the chromosome to betested. They describe tests where DNA was amplified from amniotic orchorionic villus samples. The authors suggest that the STR markers willgive PCR products of different size, and these size differences may bestudied by analyzing peak sizes in electrophoresis. It is also proposedthat quantitative real time PCR may be used to diagnose Down Syndrome bycomparing the amount of a gene located on chromosome 12 to the amount ofa gene located on another autosomal chromosome. If the ratio of thesetwo genes is 1:1, the fetus is normal, but if the ratio of these genesis 3:2, it indicates Down Syndrome. The authors propose the use of DownSyndrome marker DSCR3. They also suggest that the housekeeping geneGAPDH on chromosome 12 can be used as a reference.

Poon et al., “Differential DNA Methylation between Fetus and Mother as aStrategy for Detecting Fetal DNA in Maternal Plasma,” Clin. Chem. 48(1):35-41 discloses the detection of genes or mutations in a fetus where thesame mutation or condition is also present in maternal DNA. That is, theuse of fetal DNA in maternal plasma is limited due to the low amount offetal DNA compared to maternal DNA. The authors overcame this limitationby detecting the IGF2-H19 locus, which is maintained in a methylated DNAstatus in the paternal allele and is unmethylated in the maternalallele. The authors used a bisulfite modification kit wherebyunmethylated cytosine residues were converted to uracil. The sequencedifference between methylated and unmethylated DNA sequences could bedistinguished with different PCR primers. DNA extracted from buffy coatwas used.

Science 309:1476 (2 Sep. 2005) News Focus “An Earlier Look at Baby'sGenes” describes attempts to develop tests for Down Syndrome usingmaternal blood. Early attempts to detect Down Syndrome using fetal cellsfrom maternal blood were called “just modestly encouraging.” The reportalso describes work by Dennis Lo to detect the Rh gene in a fetus whereit is absent in the mother. Other mutations passed on from the fatherhave reportedly been detected as well, such as cystic fibrosis,beta-thalassemia, a type of dwarfism and Huntington's disease. However,these results have not always been reproducible.

United States Patent Application 20040137470 to Dhallan, Ravinder S,published Jul. 15, 2004, entitled “Methods for detection of geneticdisorders,” describes a method for detecting genetic disorders using PCRof known template DNA and restriction analysis. Also described is anenrichment procedure for fetal DNA. It also describes a method used todetect mutations, and chromosomal abnormalities including but notlimited to translocation, transversion, monosomy, trisomy, and otheraneuploidies, deletion, addition, amplification, fragment,translocation, and rearrangement. Numerous abnormalities can be detectedsimultaneously. The method is said to provide a non-invasive method todetermine the sequence of fetal DNA from a tissue, such as blood, drawnfrom a pregnant female, and a method for isolating free nucleic acidfrom a sample containing nucleic acid.

BRIEF SUMMARY OF THE INVENTION

The following brief summary is not intended to include all features andaspects of the present invention, nor does it imply that the inventionmust include all features and aspects discussed in this summary.

Briefly, the present invention is directed to a method of differentialdetection of target sequences in a mixture of maternal and fetal geneticmaterial. One obtains maternal tissue containing both maternal and fetalgenetic material. Preferably, the maternal tissue is maternal peripheralblood or blood plasma. The term “plasma” may include plasma or serum.The genetic material may be genomic DNA or RNA, preferably mRNA. In thecase of mRNA, one may choose target sequences corresponding to genesthat are highly expressed in the placenta for fetal genetic material.The genetic material (e.g., DNA) in each reaction sample is detectedwith a sequence specific reactant directed to at least one of two targetsequences in the genetic material to obtain a detectible reactionproduct if the target sequence is present in the reaction sample. Forexample, a probe specific to chromosome 21 is bound to the reactionsample, along with a control probe specific to another chromosome. Inmost cases, the results will be from maternal DNA, but a small number ofresults will be obtained from fetal DNA. In order to distinguish randomvariation from fetal results, a large number of reactions are run, andstatistical methods are applied to the results. The labeling anddetection in the present method is used to distinguish the presence orabsence of a single target sequence, referred to as “digital analysis,”although it may be performed with sensitive nucleic acid detectionmethods which distinguish between one and more than one target sequencein a discrete sample. Many fluorescent techniques have this sensitivity.The target sequences are chosen so that a maternal sequence and a fetalsequence are distinguishable, such as two copies of a maternal sequenceversus two copies of a fetal sequence.

The genetic material thus obtained is distributed into discrete samples,where each sample will contain, on average not more than about onetarget sequence per sample. The average of one target sequence meansthat, for practical reasons, the sample will contain, preferably 0.1 to0.8 genome equivalents per discrete sample, ideally 0.5 genomeequivalent per sample. The method may be performed with dilutionswhereby more target sequences are detected in samples containing atrisomic or increased copy number of target sequence. That is, if one isanalyzing chromosome 21, the mixture may be diluted such that, onaverage, one may detect two chromosomes present in a maternal DNA, andthree chromosomes in a Down Syndrome fetal DNA. Alternatively, themethod may be performed with dilutions whereby more reaction samples arepositive in this situation. The presence or absence of different targetsequences in the discrete samples is detected; and the results areanalyzed whereby the number of results from the discrete samples willprovide data sufficient to obtain results distinguishing differenttarget sequences. In one aspect, the method involves an analysis of atrisomy. In this method, one of the different target sequences (e.g.chromosome 21) is diploid in maternal genetic material and aneuploid infetal genetic material and another of the different target sequences(e.g. chromosome 12) is diploid in both maternal and fetal geneticmaterial.

The discrete samples are in reaction samples where the target sequencescan be analyzed. The reaction samples may be, for example, wells in amicrotiter plate, aqueous phases in an emulsion, areas in an arraysurface, or reaction chambers in a microfluidic device. The reactionsamples may be used for PCR analysis of the discrete samples. Thediscrete samples are contacted with a plurality of PCR primers,including at least one (or one forward and one reverse) primer directedspecifically to a maternal control sequence, expected to be the same inboth mother and fetus. PCR primers are also directed specifically to afetal sequence, i.e. one which may be present in both mother and fetus,but is amplified or altered in the fetus. PCR amplification will allowdetection of these two different sequences, and, according to thepresent method, there will be a differential in the case of an abnormalfetal target sequence. The PCR method may be (but is not necessarily)quantitative. Quantitative real time PCR, which includes hybridizingtarget sequences with a nucleic acid having a fluorescent label, may beused. A fluorescent probe hybridizing to the target sequence may also beused. A number of “digital PCR” protocols are known for this purpose, aswell as bead-based or emulsion PCR. While florescent probes are readilyavailable and may be used to provide sensitive results, e.g., in FRETcombinations, other labeling techniques may be used.

The number of discrete samples is chosen according to the resultsdesired. In one aspect, it is preferred that a high degree ofstatistical significance is obtained, and the number of samples is atleast about 10,000. In order to improve statistical confidence, it ispreferable to employ large numbers of reactions, preferably between 500and 100,000, more preferably between 10,000 and 100,000 or morereactions, depending on the percentage of fetal DNA present in themixture. The results to be obtained should be statistically significantfor purposes of the analysis conducted, e.g. initial screening, primarydiagnosis, etc. A commonly used measure of statistical significance whena highly significant result is desired is p<0.01, i.e., a 99% confidenceinterval based on a chi-square or t-test.

However, as shown below, results can be obtained with less, e.g. on theorder of about 500 samples, placed in separate reaction samples. Fewerdiscrete samples may be analyzed where the genetic material is presentin a higher concentration in the mixture. The mixture may be enrichedfor fetal genetic material. One method to enrich plasma DNA for fetalDNA is size separation, whereby a preparation comprising only DNAfragments less than about 300 bp are used for measuring targetsequences.

A variety of genetic abnormalities may be detected according to thepresent method, including known alterations in one or more of the genes:CFTR, Factor VIII (F8 gene), beta globin, hemachromatosis, G6PD,neurofibromatosis, GAPDH, beta amyloid, and pyruvate kinase. Thesequences and common mutations of these genes are known. Other geneticabnormalities may be detected, such as those involving a sequence whichis deleted in a human chromosome, is moved in a translocation orinversion, or is duplicated in a chromosome duplication, wherein saidsequence is characterized in a known genetic disorder in the fetalgenetic material not present in the maternal genetic material. Forexample chromosome trisomies may include partial, mosaic, ring, 18, 14,13, 8, 6, 4 etc. A listing of known abnormalities may be found in theOMIM Morbid map, http://www.ncbi.nlm.nih.gov/Omim/getmorbid.cgi.

In general, the term “aneuploidy” is used to refer to the occurrence ofone or more extra or missing chromosomes.

In one aspect, the present method of differential detection of targetsequences may involve direct sequencing of target sequences the geneticmaterial. Single molecule sequencing, as is known, is further describedbelow. The method may also comprise sequencing of amplified derivativesof the target sequences clones or amplicons of the genetic material.That is, a target sequence in a discrete sample is amplified by PCR,i.e. as an amplicon, or cloned into a vector that is grown up andthereby amplified by obtaining multiple copies of the vector insert.

In another aspect, the present invention comprises materials selectedand combined for carrying out the present methods. Thus is provided akit for differential detection of target sequences in maternal and fetalDNA in a mixed DNA sample, comprising primers specific for a geneticallyabnormal sequence and a control sequence, such as two chromosomes, oneof which is possibly aneuploid and one of which is presumed diploid; aPCR reaction buffer for forming a PCR reaction sample with the primersin a device having separate reaction samples; and a size separationmedium for separating the DNA sample into a fraction having less thanabout 1000 bp. The size separation medium may be gel or centrifugationmaterial for recovering smaller DNA fragments and thus enriching fetalDNA. The kit may further comprise a pair of primers specific tochromosome 21. The kit may further comprise the device having separatereaction samples for discrete samples. The device may be a microfluidicdevice or a microtiter plate having at least 1,000 discrete reactionsamples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the present analytical method,showing distribution of genetic material into compartments (1A),chromosome peaks of different height (1B), and statistical analysis ofchromosomes (1C);

FIG. 2 is a photograph of a microfluidic chip having 12 panels (numbered1-12) containing DNA with chromosome 21 labeled;

FIG. 3 is a photograph of a microfluidic chip having 12 panels (numbered1-12) containing DNA with chromosome 12 labeled; and

FIG. 4 is a graph showing results from experiments done using digitalanalysis of mixed normal and trisomic (Down Syndrome, trisomy 21) DNA.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Outline I. Overview II.Description of Steps

A. Tissue Preparation

B. Distribution of DNA molecules

C. Detection and Quantification

-   -   1. Digital PCR Methods    -   2. Bead emulsion PCR    -   3. Microfluidic Dilution with PCR    -   4. Single molecule detection and/or sequencing

D. Quantitative evaluation

III. Specific applications

A. Preparation for trisomy with frequency analysis.

B Sample Protocol

IV. Examples I. OVERVIEW

The methods and materials described below apply techniques for analyzingnumerous nucleic acids contained in a tissue sample (preferably serumor, more preferably, plasma) containing a mixture of DNA from both themother and the fetus, and allowing detection of small but statisticallysignificant differences.

The present invention involves the analysis of maternal blood for agenetic condition, wherein the mixed fetal and maternal DNA in thematernal blood is analyzed to distinguish a fetal mutation or geneticabnormality from the background of the maternal DNA. It has been foundthat, using a combination of steps, a DNA sample containing DNA fromboth the mother and the fetus can be analyzed to distinguish a geneticcondition present in a minor fraction of the DNA, which represents thefetal DNA. The method employs “digital analysis,” in which the DNA inthe sample is isolated to a nominal single target molecule in a smallreaction volume. Each sample mixture has a possibility of havingdistributed in it less than 1 target (i.e., 0 target) or more than onetarget. Next, the target molecules are detected in each reaction well,preferably as target sequences which are amplified, which may include aquantization of starting copy number of the target sequence, that is, 0,1, 2, 3, etc. A control sequence is used to distinguish an abnormalincrease in the target sequence, e.g., a trisonomy. Thus there is adifferential detection of target sequences, one of which is chosen torepresent a normal genotype present in both mother and offspring, andone of which is chosen for detection of an abnormal genotype in theoffspring, where the target sequence in the offspring will be differentfrom that of the mother, e.g. in trisomy.

FIG. 1A illustrates an embodiment where quantitative detection, e.g.quantitative real time PCR, is used. Blood 10 is processed to obtainplasma DNA 12, which is diluted and distributed into aliquots 14. Theseare added to reactions wells 1A through 5D. Shown in the wells aretargets representing chromosomes 21 and 22. In well 2A, no target DNA isfound; some wells (not shown) may have excess DNA. In well 3B, fetal DNAhaving trisomy 21 (Down Syndrome) is found. The remainder of the wellscontains maternal DNA. The DNA is amplified and/or labeled and aquantitative readout is obtained, as shown at 16. Peak 18 representingwell 3B will be 50% higher than the peaks from the other well, or thepeaks from a reference sequence on chromosome 22. Well A2, lackingeither 21 or 22, will have no peak. The peaks are shown at 20. A singlerun will have numerous random variations, such as wells that have notarget sequence, or have duplication through sample variability. Also,samples with no target will clearly result in no peak at all; wells withtwo or more targets, will give peaks significantly higher than peak 18,i.e., 2× or 2.5× controls. These results are distinguished by running amultitude of reactions, followed by statistical analysis that candiscriminate random variations from true results.

FIG. 1C illustrates an embodiment where the DNA is distributed in a moredilute fashion (less than 1, or about one half genome equivalents perwell). In this case chromosome 21 labels (primers) will generate morepositives than chromosome 22 (a diploid chromosome) specific labels(e.g., primers) due simply to the slightly greater abundance ofchromosome 21 in a trisomy-containing sample. As shown, some wells willcontain positives 20 for both chromosomes, some will contain negatives22 for both chromosomes, but some will contain blanks 24 for the diploidchromosome and peaks for the trisomic chromosome, due to its greaterabundance. The data from a higher peak 18 is not used in this mode. Asexplained below, this slight difference can be made statisticallysignificant by examining a large number of wells, and by the sensitivityof the present method to a single molecule.

Thus, the present method comprises generally the following steps:

1. Obtaining a tissue containing DNA from a pregnant subject, which DNAis known to have about 3% fetal DNA. This material is preferably drawnblood, and the circulating DNA is found in the blood plasma, rather thanin cells. The blood or plasma may optionally be enriched for fetal DNAby known methods, such as size fractionation to select for DNA fragmentsless than about 300 bp. Alternatively, maternal DNA, which tends to belarger than about 500 bp may be excluded. Another enrichment step may beto treat the blood sample with formaldehyde, as described in Dhallan etal. “Methods to Increase the Percentage of Free Fetal DNA Recovered Fromthe Maternal Circulation,” J. Am. Med. Soc. 291(9): 1114-1119 (March2004).2. Distributing single DNA molecules from this sample to a number ofdiscrete reaction samples, where the number of reaction samples isselected to give a statistically significant result for the number ofcopies of a target in the DNA molecules. Further, the reaction sample isconfined to a small volume to bring the reaction molecules into closeapproximation. The amount of DNA molecule per reaction sample ispreferably on the order of one copy of the chromosome of interestequivalent per reaction sample.3. Detecting the presence of the target in the DNA in a large number ofreaction samples, preferably with a sequence specific technique such ashighly multiplexed short read sequencing or a PCR reaction wherein thePCR product is labeled to give a convenient quantitative read out. Thedetection step is referred to here as “digital PCR” and may be carriedout by a variety of methods, such as (a) by PCR on samples diluted intoindividual wells of a microtiter plate; (b) PCR on samples diluted intoemulsions containing primers immobilized to beads; or (c) PCR on samplestrapped in a microfluidic chamber; and4. Quantitative analysis of the detection of the maternal and fetaltarget sequences. In some cases this may include targets to differentregions, such as probes to a target on a chromosome suspected of beingpresent in an abnormal copy number (trisonomy) compared to a normaldiploid chromosome, which is used as a control.

II. DESCRIPTION OF STEPS A. Tissue Preparation

The present method is directed to non-invasive testing. The preferredstarting material is maternal peripheral venous blood. In order toobtain sufficient DNA for testing, it is preferred that 10-20 mL ofblood be drawn, in order to obtain about at least 10,000 genomeequivalents of total DNA. This sample size is based on an estimate offetal DNA being present as roughly 25 genome equivalents/mL of maternalplasma in early pregnancy, and a fetal DNA concentration of about 3.4%of total plasma DNA. However, less blood may be drawn for a geneticscreen where less statistical significance is required, or the DNAsample is enriched for fetal DNA.

It should be noted that, while the present description refers throughoutto DNA, fetal RNA found in maternal blood may be analyzed as well. Asdescribed in Ng et al., “mRNA of placental origin is readily detectablein maternal plasma,” Proc. Nat. Acad. Sci. 100(8): 4748-4753 (2003), hPL(human placental lactogen) and hCG (human chorionic gonadotropin) mRNAtranscripts were detectable in maternal plasma, as analyzed using therespective real-time RT-PCR assays. In the present method, mRNA encodinggenes expressed in the placenta and present on the chromosome ofinterest are used. For example, DSCR4 (Down syndrome critical region 4)is found on chromosome 21 and is mainly expressed in the placenta. ItsmRNA sequence may be found at GenBank NM_(—)005867. In this case, it ispreferred to use RNase H minus (RNase H—) reverse transcriptases (RTs)to prepare cDNA for detection. RNase H—RTs are available from severalmanufacturers, with SuperScript™ II (Invitrogen) being the most widelyused. Reverse transcriptase PCR may be used as described below forchromosomal DNA.

i. Enrichment of DNA or RNA from Plasma

The maternal blood may be processed to enrich the fetal DNAconcentration in the total DNA, as described in Li et al., supra.Briefly, circulatory DNA is extracted from 5- to 10-mL maternal plasmausing commercial column technology (Roche High Pure Template DNAPurification Kit; Roche, Basel, Switzerland) in combination with avacuum pump. After extraction, the DNA is separated by agarose gel (1%)electrophoresis (Invitrogen, Basel, Switzerland), and the gel fractioncontaining circulatory DNA with a size of approximately 300 bp iscarefully excised. The DNA is extracted from this gel slice by using anextraction kit (QIAEX II Gel Extraction Kit; Qiagen, Basel, Switzerland)and eluted into a final volume of 40-μL sterile 10-mM trishydrochloricacid, pH 8.0 (Roche).

DNA may be concentrated by known methods, including centrifugation andvarious enzyme inhibitors. The DNA is bound to a selective membrane(e.g., silica) to separate it from contaminants. The DNA is preferablyenriched for fragments circulating in the plasma, which are less than1000 base pairs in length, generally less than 300 bp. This sizeselection is done on a DNA size separation medium, such as anelectrophoretic gel or chromatography material. Such a material isdescribed in Huber et al., “High-resolution liquid chromatography of DNAfragments on non-porous poly(styrene-divinylbenzene) particles,” NucleicAcids Res. 1993 March 11; 21(5): 1061-1066, gel filtrationchromatography, TSK gel, as described in Kato et al., “A New Packing forSeparation of DNA Restriction Fragments by High Performance LiquidChromatography,” J. Biochem, 1984, Vol. 95, No. 1 83-86.

In addition, enrichment may be accomplished by suppression of certainalleles through the use of peptide nucleic acids (PNAs), which bind totheir complementary target sequences, but do not amplify.

Plasma RNA extraction is described in Enders et al., “The Concentrationof Circulating Corticotropin-releasing Hormone mRNA in Maternal PlasmaIs Increased in Preeclampsia,” Clinical Chemistry 49: 727-731, 2003. Asdescribed there, plasma harvested after centrifugation steps is mixedTrizol LS reagent (Invitrogen) and chloroform. The mixture iscentrifuged, and the aqueous layer transferred to new tubes. Ethanol isadded to the aqueous layer. The mixture is then applied to an RNeasymini column (Qiagen) and processed according to the manufacturer'srecommendations.

ii. Blood—Extraction from Fetal Cells

United States Patent Application 20040137470 to Dhallan, Ravinder S,published Jul. 15, 2004, entitled “Methods for detection of geneticdisorders,” describes an enrichment procedure for fetal DNA,” in whichblood is collected into 9 ml EDTA Vacuette tubes (catalog numberNC9897284) and 0.225 ml of 10% neutral buffered solution containingformaldehyde (4% w/v), is added to each tube, and each tube gently isinverted. The tubes are stored at 4° C. until ready for processing.

Agents that impede cell lysis or stabilize cell membranes can be addedto the tubes including but not limited to formaldehyde, and derivativesof formaldehyde, formalin, glutaraldehyde, and derivatives ofglutaraldehyde, crosslinkers, primary amine reactive crosslinkers,sulfhydryl reactive crosslinkers, sulfhydryl addition or disulfidereduction, carbohydrate reactive crosslinkers, carboxyl reactivecrosslinkers, photoreactive crosslinkers, cleavable crosslinkers, etc.Any concentration of agent that stabilizes cell membranes or impedescell lysis can be added. In a preferred embodiment, the agent thatstabilizes cell membranes or impedes cell lysis is added at aconcentration that does not impede or hinder subsequent reactions.

Flow cytometry techniques can also be used to enrich fetal cells(Herzenberg et al., PNAS 76: 1453-1455 (1979); Bianchi et al., PNAS 87:3279-3283 (1990); Bruch et al., Prenatal Diagnosis 11: 787-798 (1991)).U.S. Pat. No. 5,432,054 also describes a technique for separation offetal nucleated red blood cells, using a tube having a wide top and anarrow, capillary bottom made of polyethylene. Centrifugation using avariable speed program results in a stacking of red blood cells in thecapillary based on the density of the molecules. The density fractioncontaining low-density red blood cells, including fetal red blood cells,is recovered and then differentially hemolyzed to preferentially destroymaternal red blood cells. A density gradient in a hypertonic medium isused to separate red blood cells, now enriched in the fetal red bloodcells from lymphocytes and ruptured maternal cells. The use of ahypertonic solution shrinks the red blood cells, which increases theirdensity, and facilitates purification from the more dense lymphocytes.After the fetal cells have been isolated, fetal DNA can be purifiedusing standard techniques in the art.

Further, an agent that stabilizes cell membranes may be added to thematernal blood to reduce maternal cell lysis including but not limitedto aldehydes, urea formaldehyde, phenol formaldehyde, DMAE(dimethylaminoethanol), cholesterol, cholesterol derivatives, highconcentrations of magnesium, vitamin E, and vitamin E derivatives,calcium, calcium gluconate, taurine, niacin, hydroxylamine derivatives,bimoclomol, sucrose, astaxanthin, glucose, amitriptyline, isomer Ahopane tetral phenylacetate, isomer B hopane tetral phenylacetate,citicoline, inositol, vitamin B, vitamin B complex, cholesterolhemisuccinate, sorbitol, calcium, coenzyme Q, ubiquinone, vitamin K,vitamin K complex, menaquinone, zonegran, zinc, ginkgo biloba extract,diphenylhydantoin, perftoran, polyvinylpyrrolidone, phosphatidylserine,tegretol, PABA, disodium cromglycate, nedocromil sodium, phenyloin, zinccitrate, mexitil, dilantin, sodium hyaluronate, or polaxamer 188.

An example of a protocol for using this agent is as follows: The bloodis stored at 4° C. until processing. The tubes are spun at 1000 rpm forten minutes in a centrifuge with braking power set at zero. The tubesare spun a second time at 1000 rpm for ten minutes. The supernatant (theplasma) of each sample is transferred to a new tube and spun at 3000 rpmfor ten minutes with the brake set at zero. The supernatant istransferred to a new tube and stored at −80° C. Approximately twomilliliters of the “buffy coat,” which contains maternal cells, isplaced into a separate tube and stored at −80° C.

iii. Plasma-Free Fetal DNA

Genomic DNA may be isolated from the plasma using the Qiagen Midi Kitfor purification of DNA from blood cells, following the manufacturer'sinstructions (QIAmp DNA Blood Midi Kit, Catalog number 51183). DNA iseluted in 100 mu.l of distilled water. The Qiagen Midi Kit also is usedto isolate DNA from the maternal cells contained in the “buffy coat.”

Finally, it is noted that, in certain embodiments, one may also usesamples from tissue, saliva, urine, tear, vaginal secretion, breastfluid, breast milk, or sweat.

B. Distribution of DNA Molecules

In the illustrated method, the genomic DNA obtained from a maternaltissue as described above is diluted into multiple reaction samples,e.g. in multiwell plates, so that there is, on average, less than onegenome equivalent per well. Thus, when the individual discrete samplesare analyzed for the presence of the genetic abnormality to be tested,the DNA (chromosome) to be analyzed will, on average, either be presentor absent, permitting so-called “digital analysis.” A reaction sample ingeneral will contain a single template molecule (haplotype), two targetmolecules (diploid) or three target molecules (trisomy).

The wells provide discrete reaction samples and may be implemented in anumber of devices, such as a microtiter plates, beads in an emulsion, ora microfluidic device. These are described in detail below. The devicemust be capable of carrying out a large number of discrete amplificationreactions. As described below, this number should be, at a minimum,10,000 reactions, and preferably on the order of 100,000 reactions. Thereaction sample is preferably holds about 10-100 μL of a PCR reactionsample containing the genomic DNA, nucleotides (dNTPs), polymerase andappropriate PCR primers. The primers are used in conjunction with alabel for rapid quantitative detection of PCR products. The type oflabeling will depend on the amplification/detection system used, e.g., a“molecular beacon” fluorescent probe for microtiter plate basedamplification. This type of probe is described, for example, inVogelstein et al, supra. Alternatively, labeling may be done with SYBRGreen, which has very low fluorescence in the absence of double strandedDNA and very high fluorescence in the presence of double stranded DNA.

Another form of parallel analysis useful in the present invention issingle molecule analysis. Again, a sample is diluted to contain lessthan a nominal single genome equivalent of DNA, and the presence of thetarget of interest (i.e., chromosome 21 trisomy) can be determined in alarge number of samples. By analyzing a large number of samples, thefetal DNA can be distinguished from the maternal DNA. This is termed a“digital analysis,” because each well will have, on average, one genomeequivalent per cell, and furthermore, the dilution may be read as abinary “yes-no” result as to the presence of the chromosome or othersequence to be counted.

Another method for single molecule analysis involves the use ofsite-specific fluorescent tags that are detected as the DNA is drawnthrough a microfluidic device in a single molecule, elongated flow. Anexample of this technique, described below, is termed “direct linearanalysis,” or DLA.

C. Detection and Quantification

Having isolated the sample DNA into a nominal genome equivalent, thepresence of the DNA sequence or chromosome of interest must bequantified. This may be done either in single molecule mode, or with anamplified product.

While the preferred embodiment of the invention is described in terms ofPCR, the invention is primarily directed to the use of multipleindividual genetic sequence detections. In some embodiments, the methodof amplification maybe, for example, self-sustained sequence reaction,ligase chain reaction, rapid amplification of cDNA ends, polymerasechain reaction and ligase chain reaction, Q-beta phage amplification,strand displacement amplification, or splice overlap extensionpolymerase chain reaction.

Also, while detection may be conveniently be carried out by a sequencespecific probe, detection may also be carried out by directly sequencinga region of interest to determine if it is the target sequence ofinterest.

1. Digital PCR Methods

While the presently known PCR methods may be multiplexed, that is, runwith multiple primers to multiple targets, it is preferred to limit thenumber of primer pairs in a given reaction. Generally, there will be twoprimer pairs: one for amplifying a test sequence, and another pair foramplifying a control sequence. Primers are designed according to knownparameters for avoiding secondary structures and self-hybridization.Further, both primer pairs should anneal and melt at about the sametemperatures.

Primers

Primers can be prepared by a variety of methods including but notlimited to cloning of appropriate sequences and direct chemicalsynthesis using methods well known in the art (Narang et al., MethodsEnzymol. 68:90 (1979); Brown et al., Methods Enzymol. 68:109 (1979)).Primers can also be obtained from commercial sources such as OperonTechnologies, Amersham Pharmacia Biotech, Sigma, and Life Technologies.The primers can have an identical melting temperature. The lengths ofthe primers can be extended or shortened at the 5′ end or the 3′ end toproduce primers with desired melting temperatures. Also, the annealingposition of each primer pair can be designed such that the sequence and,length of the primer pairs yield the desired melting temperature. Thesimplest equation for determining the melting temperature of primerssmaller than 25 base pairs is the Wallace Rule (Td=2(A+T)+4(G+C)).Computer programs can also be used to design primers, including but notlimited to Array Designer Software (Arrayit Inc.), Oligonucleotide ProbeSequence Design Software for Genetic Analysis (Olympus Optical Co.),NetPrimer, and DNAsis from Hitachi Software Engineering. The TM (meltingor annealing temperature) of each primer is calculated using softwareprograms such as Oligo Design, available from Invitrogen Corp.

The annealing temperature of the primers can be recalculated andincreased after any cycle of amplification, including but not limited tocycle 1, 2, 3, 4, 5, cycles 6-10, cycles 10-15, cycles 15-20, cycles20-25, cycles 25-30, cycles 30-35, or cycles 35-40. After the initialcycles of amplification, the 5′ half of the primers is incorporated intothe products from each loci of interest, thus the TM can be recalculatedbased on both the sequences of the 5′ half and the 3′ half of eachprimer. Any DNA polymerase that catalyzes primer extension can be usedincluding but not limited to E. coli DNA polymerase, Klenow fragment ofE. coli DNA polymerase 1, T7 DNA polymerase, T4 DNA polymerase, Taqpolymerase, Pfu DNA polymerase, Vent DNA polymerase, bacteriophage 29,REDTaq™ Genomic DNA polymerase, or sequenase. Preferably, a thermostableDNA polymerase is used. A “hot start” PCR can also be performed whereinthe reaction is heated to 95° C. for two minutes prior to addition ofthe polymerase or the polymerase can be kept inactive until the firstheating step in cycle 1. “Hot start” PCR can be used to minimizenonspecific amplification. Any number of PCR cycles can be used toamplify the DNA, including but not limited to 2, 5, 10, 15, 20, 25, 30,35, 40, or 45 cycles. In a most preferred embodiment, the number of PCRcycles performed is such that equimolar amounts of each loci of interestare produced.

A number of specific PCR primers are useful in the present process, suchas those disclosed in technical literature of Qiagen. That literaturedescribes a protocol where DNA was purified from peripheral blood andamniocyte cultures using the QIAmp DNA Blood Mini Kit. For amplificationof the amyloid gene on chromosome 21, (NCBI gene ID 473931, accessionNC_(—)006488) primer and probe sequences were:

SEQ ID NO:1: forward primer, 5′-GGG AGC TGG TAC AGA AAT GAC TTC-3′;reverse primer, SEQ ID NO:10: 5′-TTG CTC ATT GCG CTG ACA A-3′; andprobe, SEQ ID NO:2 5′-(FAM) AGC CAT CCT TCC CGG GCC TAG G (TAMRA)-3′.

For amplification of GAPDH, (GenBank locus 12p13.31-p13.1) primers andprobe were: forward primer, SEQ ID NO: 3,5′-CCC CAC ACA CAT GCA CTTACC-3′; reverse primer, SEQ ID NO: 4,5′-CCT ACT CCC AGG GCT TTG ATT-3′;and probe, SEQ ID NO: 5, 5′-(VIC) AAA GAG CTA GGA AGG ACA GGC AAC TTG GC(TAMRA)-3′. PCR was performed using the TaqMan system, with 2 μl oftemplate DNA in each 25 μl reaction and final concentrations of 300nmol/liter of each primer and 150 nmol/liter of each dual-labeled TaqManprobe. Cycling conditions were incubation at 50° C. for 2 minutes, then95° C. for 10 minutes, followed by 40 cycles of 60° C., 1 minute and 95°C., 15 seconds.

Using the above exemplary protocol, the different ratio of the amyloidgene and the GAPDH gene in karyotypically normal and trisomy 21 sampleswas clearly distinguishable in the multiplex PCR assay, as reported inthe Qiagen product literature. Assays using a dilution series of the DNAtemplate showed that the difference remained clear over a wide range oftemplate concentrations and with starting concentrations of DNA as lowas 10 mg/liter. Of course, in a maternal blood sample, the concentrationof fetal DNA would be much lower.

Fluorescent In Situ Amplification

Fluorescent probe-based technologies, which can be performed on the PCRproducts “in situ” (i.e., in the same wells), are particularly wellsuited for this application. This method is described in detail inVogelstein PNAS 96:9236, above, and Vogelstein et al. “DigitalAmplification,” U.S. Pat. No. 6,440,705, hereby incorporated byreference for its description of this amplification procedure.

The “digi-PCR” method of Vogelstein et al. is described in theabove-mentioned patent. An exemplary protocol as set forth in thatpatent is as follows: PCR is performed in 7 μl volumes in 96 wellpolypropylene PCR plates (Marsh Biomedical Products, Rochester, N.Y.).The composition of the reactions is: 67 mM Tris, pH 8.8, 16.6 mM NH₄SO₄, 6.7 mM MgCl₂, 10 mM β-mercaptoethanol, 1 mM dATP, 1 mM dCTP, 1 mMdGTP, 1 mM TTP, 6% DMSO, 1 μM primer F1, 1 μM primer R1, 0.05 units/μlPlatinum Taq polymerase (Life Technologies, Inc.), and “one-half genomeequivalent” of DNA.

To determine the amount of DNA corresponding to one-half genomeequivalent,

DNA samples are serially diluted and tested via PCR. The amount thatyielded amplification products in half the wells, usually about 1.5 pgof total DNA, is defined as “one-half genome equivalent” and used ineach well of subsequent Digital Amplification experiments. Fifty μllight mineral oil (Sigma M-3516) is added to each well and reactionsperformed in a HybAid Thermal cycler at the following temperatures:denaturation at 94° C. for one min; 60 cycles of 94° C. for 15 sec, 55°C. for 15 sec., 70° C. for 15 seconds; 70° C. for five minutes.

MB, or molecular beacon probes, which become fluorescent on binding tothe target sequence(s), as described in more detail below, may be usedas follows:

For fluorescence analysis, 3.5 μl of a solution with the followingcomposition is added to each well: 67 mM Tris, pH 8.8, 16.6 mM NH₄ SO₄,6.7 mM MgCl₂, 10 mM β.-mercaptoethanol, 1 mM dATP, 1 mM dCTP, 1 mM dGTP,1 mM TTP, 6% DMSO, 5 μM primer, 1 mM MB-GREEN, 1 mM MB-RED, 0.1 units/μlPlatinum Taq polymerase. The plates are centrifuged for 20 seconds at6000 g and fluorescence read at excitation/emission wavelengths of 485nm/530 nm for MB-GREEN and 530 nm/590 nm for MB-RED. The plates are thenplaced in a thermal cycler for asymmetric amplification at the followingtemperatures: 94° C. for one minute; 10-15 cycles of 94° C. for 15 sec,55° C. for 15 sec., 70° C. for 15 seconds; 94° C. for one minute; and60° C. for five minutes. The plates are then incubated at roomtemperature for ten to sixty minutes and fluorescence measured asdescribed above.

MB probes are oligonucleotides with stem-loop structures that contain afluorescent dye at the 5′ end and a quenching agent (Dabcyl) at the 3′end. The degree of quenching via fluorescence energy resonance transferis inversely proportional to the 6th power of the distance between theDabcyl group and the fluorescent dye. After heating and cooling, MBprobes reform a stem-loop structure, which quenches the fluorescentsignal from the dye. If a PCR product whose sequence is complementary tothe loop sequence is present during the heating/cooling cycle,hybridization of the MB to one strand of the PCR product will increasethe distance between the Dabcyl and the dye, resulting in increasedfluorescence.

The examples below use a PCR protocol, which also relies on MB typeprobes, except in connection with a microfluidic device.

The present digital PCR methods may be used with RNA as well as DNA.Isolation of plasma RNA is described below. In this case, cDNA copiesare made and then amplified by DNA polymerase-based PCR. Differentprimers may be used for cDNA synthesis. Specific templates, based ongenetic sequences in the chromosomes of interest are preferred. See,Bustina et al., “Pitfalls of Quantitative Real-TimeReverse-Transcription Polymerase Chain Reaction,” Journal ofBiomolecular Techniques, 15:155-166 (2004). Use of mRNA fromconsitutively expressed, i.e., housekeeping genes, may be used for acontrol, and genes that are highly expressed in placenta (describedbelow) are preferred. Currently four different chemistries, TaqMan®(Applied Biosystems, Foster City, Calif., USA), Molecular Beacons,Scorpions® and SYBR® Green (Molecular Probes), are available forreal-time PCR. All of these chemistries allow detection of PCR productsvia the generation of a fluorescent signal and may be adapted toreverse-transcription PCR. Ambion's MessageSensor™ RT Kit includes anRNase H+ MMLV RT. MessageSensor includes a total RNA control, a controlhuman GAPDH primer set, RNase inhibitor, and nucleotides, as well as abuffer additive that enables detection with SYBR® Green dye. Ambionrecommends using 18S rRNA as an internal control because it shows lessvariance in expression across treatment conditions than β-actin andGAPDH. A chromosome 21-encoded gene (LOC90625) which shows strongexpression in first trimester placenta similar to CSH1 (human placentallactogen) and was selected for plasma analysis in Oudejans et al.,“Detection of Chromosome 21-encoded mRNA of Placental Origin in MaternalPlasma,” Clinical Chemistry 49: 1445-1449, 2003. Specific primers foruse with this gene are given in this paper. Uniquely expressedchromosome 21 transcripts are described at Gardiner et al., “Analysis ofhuman chromosome 21: correlation of physical and cytogenetic maps; geneand CpG island distributions,” E.M.B.O.J. 9(1):25-34 (1990), namely cDNAof identified products ETS2, MX1, MX2, CBS, COL6A1 and BCEI, which canbe partially sequenced or mapped according to eh present methods.

2. Bead Emulsion PCR

Emulsion PCR has been used to prepare small beads with clonallyamplified DNA—in essence, each bead contains one type of amplicon ofdigital PCR. (Dressman et al, Proc. Natl. Acad. Sci. USA. 100, 8817(Jul. 22, 2003)). By using specific primers for regions of chromosomes Aand B while performing emulsion PCR, one will create beads with digitalamplicons from only these two chromosomes, and it is only necessary tocount the number of positive beads of each type. There are many ways todo this; we will point out two of them. First, use two different speciesof beads (either in size or fluorescent labeling) to anchor the twoamplicons respectively. Alternatively, one could label the non-anchoredprimers with different fluorophores and use a single bead type. Afteramplification, the positive beads (amplicons) of each type can becounted with methods such as flow cytometry or simply by counting themin a suitably equipped microscope.

This technique is further described in Dressman et al (supra) andDressman et al. PCT publication WO2005010145, “METHOD AND COMPOSITIONSFOR DETECTION AND ENUMERATION OF GENETIC VARIATIONS,” published 2005,Jan. 3, and hereby incorporated by reference for its description of abead-based process. Briefly, in Step 1, Magnetic beads covalently coatedwith streptavidin are bound to biotinylated oligonucleotides (“oligos”).In Step 2, an aqueous mix containing all the necessary components forPCR plus primer-bound beads and template DNA are stirred together withan oil/detergent mix to create microemulsions. The aqueous compartments(which may be illustrated as small droplets in an oil layer) contain anaverage of <1 template molecule and <1 bead. Different templates(control and test) may be pictured in one or less droplets to representtwo template molecules whose sequences differ by one or manynucleotides. In Step 3, the microemulsions are temperature cycled as ina conventional PCR. If a DNA template and a bead are present together ina single aqueous compartment, the bead bound oligonucleotides act asprimers for amplification. Then, one may picture straight linescorresponding to PCR products attached to the corresponding templatesconnected to the beads to represent extension products from the twodifferent kinds of templates. In Step 4, the emulsions are broken andthe beads are purified with a magnet. In Step 5, after denaturation, thebeads are incubated with oligonucleotides that can distinguish betweenthe sequences of the different kinds of templates. Fluorescently labeledantibodies are then used to label the bound hybridization probes. Thisrenders the beads containing PCR product as different colors (e.g., redor green) upon appropriate laser excitation. In Step 6, flow cytometryis used to count the red and green beads. Preferably each bead is boundto at least 10, 50, 100, 500, or 1000 molecules of the same nucleic acidsequence.

For purposes of detailed description, the following example is takenfrom the above-quoted PCT publication:

Detailed Exemplary Protocol Using Bead Emulsions

Step 1-Coupling oligonucleotides to beads. Superparamagnetic beads of1.05±0.1 um in diameter, covalently bound to streptavidin, are purchasedfrom Dynal Biotech, Inc. (650.01, Lake Success, N.Y.). Beads are washedonce with 1×PCR buffer (53286, Invitrogen, Carlsbad, Calif.) thensuspended in Bind and Wash Buffer (BWB) (5 mMTris-HCI, 0.5 mM EDTA, 1.0MNaCI, pH 7.5). Beads are incubated in BWB for 30 min at roomtemperature in the presence of 10 mM oligonucleotides. Theseoligonucleotides are modified with a dual biotin group at the 5′ endwith the biotin groups separated by a six-carbon linker (IDT,Coralville, Iowa). After binding, the beads are washed 3 times with1×PCR buffer to thoroughly remove unbound oligonucleotides.

Step 2-Preparing microemulsions. Microemulsions for PCR are prepared inan oil phase that is composed of 4.5% Span 80 (S6760, Sigma, St. Louis,Mo.), 0.40% Tween 80 (Sigma S-8074), and 0.05% Triton X-100 (SigmaT-9284) in mineral oil (Sigma M-3516). The aqueous phase consists of 67mMTris-HCl (pH 8.8), 16.6 mM NH4S04, 6.7 mMMgC12, 10 mM(3-mercaptoethanol, 1 mMdATP, 1 mMdCTP, 1 mMdGTP, 1 mMdTTP, 0.05 μMforward primer, 25 μM reverse primer, 45 units Platinum Taq (Invitrogen10966-034), various amounts of template DNA, and ˜108oligonucleotide-coupled beads in a total volume of 300 μl. The forwardprimer is an oligonucleotide whose sequence is identical to the 3′20-22nt of that described in step 1 and is not modified with biotin.

Water-in-oil microemulsions are prepared by drop wise addition of 200microliters of the aqueous phase to 400 microliters of the oil phasepreviously placed in a 2 ml round bottom cryogenic vial (430661,Corning, Corning, N.Y.).

The drop wise addition is performed over-one minute while the mixture isbeing stirred at 1400 RPM with a magnetic microstir bar (58948-353, VWR,Plainfield, N.J.) on a VWR model 565 Magnetic Stirrer. After theaddition of the aqueous phase, the mixture continued to be stirred for atotal time of 30 minutes. Two emulsions are made at once by placing twotubes in a rack placed at the center of the magnetic stirrer.

Step 3-PCR cycling. The emulsions are aliquotted into five wells of a 96well PCR plate, each containing 100 μl. PCR is carried out under thefollowing cycling conditions: 94° C. for 2 minutes; 40 cycles of 94° C.for 15 seconds, 57° C. for 30 seconds, 70° C. for 30 seconds. The PCRproducts analyzed in this study ranged from 189 to 239 bp.

Step 4-Magnetic capture of beads. After PCR cycling, the microemulsionfrom five wells of a PCR plate are pooled and broken by the addition 800microliters of NX buffer (100 mMNaCI containing 1% Triton X-100, 10mMTris-HCl, pH 7.5, 1 mM EDTA) in a 1.5 ml tube (Corning 430909). Aftervortexing for—20 sec. the beads are pelleted by centrifugation in amicrocentrifuge at 8000 rpm (5000 g) for 90 seconds. The top oil phase,and all but—300 microliters of the aqueous phase, is removed from thetube and 600 microliters of NX buffer is added. These steps arerepeated. The tube is then placed on a magnet (Dynal MPC-S) and the restof the supernatant is carefully pipetted off. The beads are washed anadditional 3 times with 1×PCR buffer using magnetic separation ratherthan centrifugation and finally re-suspended in 100 microliters of 1×PCRbuffer.

Step 5-Sequence differentiation. Two oligonucleotide probes are used foreach reaction. One is 5′-labeled with 6-carboxyfluorescein (6-FAM) andis specific for one allele while the second is 5′-labeled with biotinand is specific for the other allele. Probes are synthesized by IDT. The30 microliters hybridization reactions contained 10 μM of each probe and5-25 million beads inl×PCR buffer. Reactions are performed in PCR plateson a thermal cycler by heating to 94° C. for 30 seconds then cooling to75° C. at a rate of 0.5° C. per second, cooling to 45° C. at 0.2° C. persecond, and finally cooled to 30° C. at 1° C. per second.

All subsequent steps are performed at room temperature. The reactionsare transferred to a 96 well Costar plate (Corning 3797) and placed on a96 well magnet. Beads are collected magnetically by exposing them to themagnet for 2 minutes. The supernatant is removed and the beads washed 3times with 1×PCR buffer by pipetting them and collecting for twominutes. They are finally resuspended in 100 microliters B-PCR buffer (1mg/mL BSA in 1×PCR buffer).

The beads are then incubated for 10 minutes in a total volume of 100microliters B-PCR buffer containing 3 μg of Alexa-488 rabbitanti-fluorescein antibody (Molecular ProbesA-11090, Eugene, Oreg.) and 3μg of Nutravidin labeled with R-phycoerytbrin (Molecular Probes A-2660)in B-PCR buffer. The beads are washed three times and resuspended inB-PCR buffer as described above. They are then incubated for ten minutesin a total volume of 100 microliters B-PCR buffer containing 6 μg ofAlexa 488-conjugated chicken anti-rabbit antibody (Molecular ProbesA-21441) and 3 μg of biotinylated goat anti-avidin antibody (BA-0300,Vector Laboratories, Burlingame, Calif.). The beads are washed threetimes and resuspended in B-PCR buffer as described above. They are thenincubated for ten minutes in a total volume of 100 microliters B-PCRbuffer containing 3 μg of an Alexa 488-conjugated goat anti-chickenantibody (Molecular Probes A-11039) and 3 micrograms ofR-phycoerytbrin-labeled streptavidin (Molecular Probes S-866). Thissolution is then washed an additional 3 times with 1×PCR buffer andresuspended in 20 microliters of 1×PCR buffer.

Step 6-Flow Cytometry. The bead suspension is diluted to a concentrationof—106-107 beads per ml in 10 mMTris-HCI, 1 mMEDTA (351-010-131, QualityBiological, Inc., Gaithersburg, Md.) and analyzed using a LSR instrument(BD Biosciences, Franklin Lakes, N.J.). The instrument is set up forstandard two-color analysis using an argon laser and optical filtersthat distinguished between the two fluorescent dyes. No spectraldeconvolution is required as the major bead populations are wellseparated. In some cases, scanning is performed with FACScan orFACSCalibur instruments (BD Biosciences).

3. Microfluidic Dilution with PCR

Another approach to digital PCR involves the use of microfluidics toachieve the digital PCR conditions used in the present method.

Generally, a DNA sample obtained as described above is diluted into anappropriate concentration, mixed with PCR reagents, primers, dNTPs, etc.and flowed through a number of channels which may be closed off inmultiple segments, resulting in a number of discrete reaction samples,or chambers. The chambers may be subjected to PCR thermal cycling andthe products quantitatively detected by florescence, as described above.

A suitable microfluidic device is produced by Fluidigm Corporation,termed the Digital Isolation and Detection IFC (integrated fluidcircuit). A suitable device is also described in U.S. Pat. No. 6,960,437to Enzelberger, et al., issued Nov. 1, 2005 entitled “Nucleic acidamplification utilizing microfluidic devices,” hereby incorporated byreference for purposes of describing a microfluidic device capable ofsupporting multiple parallel nucleic acid amplifications and detections.As described in this patent, one exemplary microfluidic device forconducting thermal cycling reactions includes in the layer with the flowchannels a plurality of sample inputs, a mixing T-junction, a centralcirculation loop (i.e., the substantially circular flow channel), and anoutput channel. The intersection of a control channel with a flowchannel can form a microvalve. This is so because the control and flowchannels are separated by a thin elastomeric membrane that can bedeflected into the flow channel or retracted therefrom. Deflection orretraction of the elastomeric membrane is achieved by generating a forcethat causes the deflection or retraction to occur. In certain systems,this is accomplished by increasing or decreasing pressure in the controlchannel as compared to the flow channel with which the control channelintersects. However, a wide variety of other approaches can be utilizedto actuate the valves including various electrostatic, magnetic,electrolytic and electrokinetic approaches. Another microfluidic device,adapted to perform PCR reactions, and useful in the present methods, isdescribed in US 2005/0252773 by McBride, et al., published Nov. 17,2005, entitled “Thermal reaction device and method for using the same.”

The substantially circular central loop and the control channels thatintersect with it form the central part of the rotary pump. The pump(s)that cause solution to be flowed through the substantially circular flowchannel consist of a set of at least three control channels that areadjacent to one another and which intersect the substantially circularbranch flow channel (i.e., the central loop). When a series of on/offactuation sequences are applied to the control channels, the fluid inthe central loop can be peristaltically pumped in a chosen direction,either clockwise or counterclockwise. The peristaltic pumping actionresults from the sequential deflection of the membranes separating thecontrol channels and flow channel into or out of the flow channel. Ingeneral, the higher the actuation frequency, the faster the fluidrotates through the central loop. However, a point of saturation iseventually reached at which increased frequency does not result infaster fluid flow. This is primarily due to limitations in the rate atwhich the membrane can return to an unactuated position. One systemexemplified has two sets of pumps and (i.e., two sets of three controlchannels that overlay the substantially circular flow channel) a singlepump can be utilized (i.e., a single set of three control channelsoverlaying the substantially circular flow channel). Furthermore, whileeach pump is shown as including three control channels, more controlchannels can be utilized. It should also be understood that the threecontrol channels can be different segments of a single control channelthat overlay the flow channel.

The detailed description of multiple sample analysis being carried outin wells does not mean that the target sequences need to be physicallyseparated into wells, as the sequences may be in samples which areisolated simply by being on different beads (as described above) or byadherence to different areas of a substrate (as described below).

4. Single Molecule Detection/Sequencing Methods

It should be appreciated that methods involving PCR or otheramplification are not the only way to detect or enumerate the moleculesin a given discrete reaction sample. It is possible to use singlemolecule flow cytometry to count single molecules that have been labeledwith a sequence-specific fluorescent probe. It is also possible tosequence the target sequence in the reaction sample directly, eitherafter amplification or at the single molecule level.

Fluorescent Nucleotide Incorporation by DNA Polymerase

As described in the above-referenced PNAS publication by Braslavsky etal., DNA polymerase may be employed to image sequence information in asingle DNA template as its complementary strand is synthesized. Thenucleotides are inserted sequentially; only the time resolution todiscriminate successive incorporations is required. After eachsuccessful incorporation event, a fluorescent signal is measured andthen nulled by photobleaching. This method lends itself to massiveparallelism.

Briefly, this technique permits observations of single moleculefluorescence by a conventional microscope equipped with total internalreflection illumination, which reduces background fluorescence. Thesurface of a quartz slide is chemically treated to specifically anchorDNA templates while preventing nonspecific binding of free nucleotides,and a plastic flow cell is attached to the surface to exchangesolutions. DNA template oligonucleotides are hybridized to afluorescently labeled primer and bound to the surface via streptavidinand biotin with a surface density low enough to resolve singlemolecules. The primed templates are detected through their fluorescenttags, their locations are recorded for future reference, and the tagsare photobleached. Labeled nucleotide triphosphates and DNA polymeraseenzyme are then washed in and out of the flow cell while the knownlocations of the DNA templates are monitored for the appearance offluorescence. The technique uses a combination of evanescent wavemicroscopy and single-pair fluorescence resonance energy transfer(spFRET) to reject unwanted noise. The donor fluorophore excitesacceptors only within the Forster radius, thus effectively creating anextremely high-resolution near-field source. Because the Forster radiusof this fluorophore pair is 5 nm, the spatial resolution of this methodexceeds the diffraction limit by a factor of 50 and conventionalnear-field microscopy by an order of magnitude.

The genomic DNA from the tissue taken from the mother, i.e. the mixtureof fetal and maternal genetic material, may be distributed into discretesamples which are anchored to a surface and sequenced or monitored bylabeled probes to detect a target specific sequence, e.g., a uniqueregion of chromosome 21, e.g., AML1. Further guidance for thepreparation of chromosome 21-unique sequences may be found, for example,in Fuscoe et al., “An Efficient Method for Selecting Unique-SequenceClones from DNA Libraries and Its Application To Fluorescent Staining ofHuman Chromosome 21 Using in Situ Hybridization,” Genomics, vol. 5,1989, pp. 100-109. A methodology useful in the present inventionplatform is based on massively parallel sequencing of millions offragments using attachment of randomly fragmented genomic DNA to aplanar, optically transparent surface and solid phase amplification tocreate a high density sequencing flow cell with millions of clusters,each containing ˜1,000 copies of template per sq. cm. These templatesare sequenced using four-color DNA sequencing-by-synthesis technology.See, products offered by Illumina, Inc., San Diego Calif. Also, see US2003/0022207 to Balasubramanian, et al., published Jan. 30, 2003,entitled “Arrayed polynucleotides and their use in genome analysis.”

Sequencing may be combined with amplification-based methods in amicrofluidic chip having reaction chambers for both PCR and microscopictemplate-based sequencing. Only about 30 bp of random sequenceinformation are needed to identify a sequence as belonging to a specifichuman chromosome. Longer sequences can uniquely identify more particulartargets. An algorithm for designing unique sequences is described inYamada, et al. “PrimerStation: a highly specific multiplex genomic PCRprimer design server for the human genome,” Nucleic Acids Res., Jul. 1,2006; 34(Web Server issue): W665-W669, illustrative of software methodsthat can be used to identify a sequence in comparison to the knowngenome sequence. See, also Zhu et al., “Single molecule profiling ofalternative pre-mRNA splicing,” Science. 2003 Aug. 8; 301(5634):836-838,describing a single-molecule-based technology for studying mRNA.

Direct Linear Analysis (DLA)

Another method of determining the identity of genomic DNA from thepresent samples is termed direct linear analysis, and is described inChan et al. “DNA Mapping Using Microfluidic Stretching andSingle-Molecule Detection of Fluorescent Site-Specific Tags,” GenomeResearch 14:1137-1146 (2004). In this method, a microfluidic device isused for stretching DNA molecules in elongational flow that is coupledto a multicolor detection system capable of single-fluorophoresensitivity. Double-stranded DNA molecules are tagged atsequence-specific motif sites with fluorescent bisPNA (Peptide NucleicAcid) tags. The DNA molecules are then stretched in the microfluidicdevice and driven in a flow stream past confocal fluorescence detectors.DLA can provide the spatial locations of multiple specific sequencemotifs along individual DNA molecules, and thousands of individualmolecules can be analyzed per minute.

A microchip configuration and operating conditions may be preparedaccording to this publication that are adequate for stretching 50-kblong DNA. The chip includes a post field, a funnel with a 10:1 taperreduction ratio, a taper shape providing W(x) 1/×2 profile (W is thechannel width, and x is the coordinate along the flow direction), and a5 μm-wide interrogation channel. The interrogation channel has uniformcross-section to ensure constant solution velocity, which was 10-15μm/msec. Once inside the channel, stretched and tagged DNA moleculestravel through spots of focused laser light that excites fluorescence.Epi-illumination of the sample and confocal detection are arrangedwithin a fluorescence microscope.

The excitation laser beams are directed into the microscope objectivewith a dichroic mirror that reflects the light with 532 nm (beam ExI)and 633 nm (beams ExII and ExIII) wavelengths, but is transparent to thefluorescence emission excited by these beams. The emission is furthersplit by another dichroic mirror and bandpass filters. Fluorescenceexcited by the green laser is delivered by optical fiber to thephoton-counting avalanche photodiode (APD) for signal detection in datachannel 1. Fluorescence excited by red beams ExII and ExIII is directedto the APDs of data channels 2 and 3, respectively.

The above-described device may be configured with larger path lengths inorder to accommodate larger DNA strands, presumably up to entirechromosome lengths. The genomic sample is probed with a chromosome 21specific probe threaded through the interrogation channel, and thepresence of one or more chromosomes is detected.

D. Quantitative Evaluation

Digital PCR allows the detection of aneuploidy merely by countingtranscripts, as illustrated by the following calculation. Suppose thatfetal DNA is present in maternal blood at a fraction level of ε, andthat we are trying to discover an aneuploidy of order α relative toeuploidy e (in the example relating to detection of Down Syndrome inhumans, e=2 is euploidy and the Down Syndrome trisomy α=3). Ifchromosome A is euploid and represents an internal control, andchromosome B is aneuploid and is the target to be measured, then one canamplify representative segments from both chromosomes via digital PCR.In comparing the amplicons of each type, one expects to find that forevery e amplicons from chromosome A there are e(1−ε)+αε amplicons fromchromosome B. In the case of a trisomy and ε=3%, then for every 2amplicons from chromosome A one expects 2.03 amplicons from chromosomeB. While this difference is small, it can be measured. For example, ifone amplifies a sample from 1,000 cell equivalents, then one expects2,000 amplicons from chromosome A and 2,030 from chromosome B. Thedifference of 30 amplicons is in principle detectable.

The requisite statistical confidence to resolve the difference inproportions can be estimated as follows. There is a random statisticalvariation associated with the initial sample size, which goes roughly asthe square root of the number of samples taken. It is in fact oftendifficult to precisely start with a fixed number of cell equivalents,and in the previous example we expect statistical error of order 32amplicons (32˜square root(1,000)) for most sample preparationtechniques. This is the same size as the signal we are trying to detectand thus in practice one requires more than 1,000 cell equivalents forrobust detection. Precisely how many one requires depends on thestatistical certainty that is required. If one would like a result thatis significant to k standard deviations, then

k√N=N(e(1−ε)+αε−e)=Nε(α−e)

or N=(k/(ε(α−e)))²

Using the values of the previous example, if we require k=3 standarddeviations, then the number of amplicons N must be at least 10,000 forDown Syndrome detection. However, as discussed above, the number oftarget sequences needed for statistical sequences may be reduced byusing controls sequences, and, in addition, the sample may be enrichedfor fetal DNA.

III. SPECIFIC APPLICATIONS

The present invention is particularly adapted to detecting geneticabnormalities that involve quantitative differences between maternal andfetal genetic sequences. These genetic abnormalities include mutationsthat may be heterozygous and homozygous between maternal and fetal DNA,and to aneuploidies. For example, a missing copy of chromosome X(monosomy X) results in Turner's Syndrome, while an additional copy ofchromosome 21 results in Down Syndrome. Other diseases such as Edward'sSyndrome and Patau Syndrome are caused by an additional copy ofchromosome 18, and chromosome 13, respectively. The present method maybe used for detection of a translocation, addition, amplification,transversion, inversion, aneuploidy, polyploidy, monosomy, trisomy,trisomy 21, trisomy 13, trisomy 14, trisomy 15, trisomy 16, trisomy 18,trisomy 22, triploidy, tetraploidy, and sex chromosome abnormalitiesincluding but not limited to XO, XXY, XYY, and XXX.

Other chromosome specific primers are disclosed in United States PatentApplication 20050164241 to Hahn, Sinuhe, et al., published Jul. 28,2005, entitled “Non-invasive detection of fetal genetic traits,” herebyincorporated by reference in its entirety for describing methods ofsample preparation and certain PCR primers, described as follows:

The primers for the genes are prepared on the basis of nucleotidesequences obtained from databases such as GenBank, EMBL and the like.The names of the polymorphic primers and the sequences of the primersfor the genes will be shown for the respective chromosomes in thefollowing examples (#2, Example 1; #4, Example 6, #14, Example 9; #22,Example 2). The following genetic markers and polymorphic makers(Polymorphic STS Primer Pairs: D2S207, D2S177, D2S156 and D2S159, BIOSLaboratories, Inc.) are used to identify chromosome #2.

There are more than 1,000 chromosome 21 specific PCR primer sets listedat the NIH UniSTS web site, which can be located athttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=unists and found withthe search phrase “human[organism] AND 21[chr]”. UniSTS is acomprehensive database of sequence tagged sites (STSs) derived fromSTS-based maps and other experiments. STSs are defined by PCR primerpairs and are associated with additional information such as genomicposition, genes, and sequences. Similarly, primer sequences for otherhuman chromosomes can be found by appropriately modifying the searchquery.

Examples of diseases where the target sequence may exist in one copy inthe maternal DNA (heterozygous) but cause disease in a fetus(homozygous), include sickle cell anemia, cystic fibrosis, hemophilia,and Tay Sachs disease. Accordingly, using the methods described here,one may distinguish genomes with one mutation from genomes with twomutations.

Sickle-cell anemia is an autosomal recessive disease. Nine-percent of USblacks are heterozygous, while 0.2% are homozygous recessive. Therecessive allele causes a single amino acid substitution in the betachains of hemoglobin.

Tay-Sachs Disease is an autosomal recessive resulting in degeneration ofthe nervous system. Symptoms manifest after birth. Children homozygousrecessive for this allele rarely survive past five years of age.Sufferers lack the ability to make the enzyme N-acetyl-hexosaminidase,which breaks down the GM2 ganglioside lipid.

Another example is phenylketonuria (PKU), a recessively inheriteddisorder whose sufferers lack the ability to synthesize an enzyme toconvert the amino acid phenylalanine into tyrosine Individualshomozygous recessive for this allele have a buildup of phenylalanine andabnormal breakdown products in the urine and blood.

Hemophilia is a group of diseases in which blood does not clot normally.Factors in blood are involved in clotting. Hemophiliacs lacking thenormal Factor VIII are said to have Hemophilia A, and those who lackFactor IX have hemophilia B. These genes are carried on the Xchromosome, so primers and probes may be used in the present method todetect whether or not a fetus inherited the mother's defective Xchromosome, or the father's normal allele.

A listing of gene mutations for which the present method may be adaptedis found at http://www.gdb.org/gdb, The GDB Human Genome Database, TheOfficial World-Wide Database for the Annotation of the Human GenomeHosted by RTI International, North Carolina USA.

A. Preparation for Trisomy with Frequency Analysis

In this protocol, the number of positive reaction samples is used,disregarding increased intensity from three versus two chromosomes in areaction sample. That is, as described above, trisomy can be detectedeither by looking for an increased signal from a single well havingmultiple chromosomal DNA copies, or by diluting a sample and countingthe frequency of responses of the trisomic marker versus a controldiploid marker.

Fetal DNA circulating in maternal plasma is here used to providesufficient material for chromosomal analysis. DNA is extracted from ablood sample and aliquotted to different reaction chambers on the basisof genome equivalents, i.e., the entire genomic content of a singlenormal cell (46 chromosomes). This weighs about 6.6 pg. The term“nominal genome equivalent” is used to refer to the calculateddistribution of sample DNA based on a calculated genome size and DNAweight. In practice, there will be some experimental variation in DNAsample size, and, due to random fragment distribution, a given genomeequivalent will not contain exactly the DNA fragments corresponding onlyto a single complete diploid genome, but a large number, on average,will.

For each panel on the Digital Array chip, 10 ul of reaction mix isrequired. To achieve ˜⅓ panel filled, the required final concentrationof template in reaction mix should be approximately 48 copies/μl (every0.33 template per 7 nl chamber). Thus for a 10 μl reaction volume (1panel), 480 copies (˜240 genome equivalents, “GE”) of totalfree-floating DNA is required. These calculations are based on Chiu etal., “Effects of Blood-Processing Protocols on Fetal and Total DNAQuantification in Maternal Plasma,” Clinical Chemistry, 47:9. 1607-1613.2001, where real time quantitative PCR was used to estimate plasma DNAisolated under different protocols, and Li Y, Zimmermann et al., “SizeSeparation of Circulatory DNA in Maternal Plasma Permits Ready Detectionof Fetal DNA Polymorphisms,” Clinical Chemistry, 50:6. 1002-1011. 2004.

Assuming 55% blood volume is plasma, one may obtain 80% recovery fromgel extraction with a DNA preparation such as with a QIAEX II kit. Ifthere is 20 ml of blood collected, the volume of plasma=20 mlblood*0.55=11 ml. The total free-floating DNA=11 ml*1000 GE/ml=11000 GE.Therefore, one may calculate the amount of DNA<300 bp, in that 11000GE*0.27=2970 GE. The amount of DNA<300 bp after recovery=2970GE*0.8=2376 GE=4752 copies.

Thus, a 20 ml blood draw should contain enough total DNA less than 300bp (which is about 85% fetal DNA) for about 10 panels, enough, as shownbelow, to achieve statistical significance.

B. Sample Protocol

The following sample protocol provides a procedure for use in preparinga sample from maternal plasma and increasing the signal from chromosome21.

Plasma Collection: collect 20 ml peripheral blood from the pregnantsubject. This is collected in 2 tubes with EDTA as anticoagulant.Process blood within 2 hours of sample collection. The blood isprocessed first by centrifugation at 1600 g for 10 min. One aliquotsplasma to polypropylene tubes (1 ml each), with care not to disturbbuffy coat layer. Next, the supernatant is microcentrifuged at 16000 g(full speed) for 10 min to remove residual maternal cells. Then, oneextracts DNA from plasma with QIAamp Blood Mini Kit (“body fluidprotocol”). 800 μl of plasma is applied per column and eluted in 40 μlbuffer.

Depending on actual DNA concentration in plasma, one may need to processall plasma in a single column (with Midi or Maxi kit) to achieve ahigher final concentration of DNA. Then, the DNA is subjected to gelelectrophoresis (Li et al Clin. Chem. 50:6 1002-1011 2004) to separatesmaller sized DNA fragments. A UV gel tray is prepared with 1% agarosegel with 0.5 mg/L ethidium bromide. 100 bp ladder and HindIII digestedLambda phage DNA is used as markers. The extracted DNA is loaded on agel; the gel is run at 80V for 1 hour. The DNA is extracted from the gelby first excising DNA<300 bp with clean razor blade. This band isrecovered with QIAEX II Gel Extraction Kit (Qiagen) and eluted 40 ul inelution buffer.

Total DNA Quantitation with Real Time PCR:

The amount of total free-floating DNA can be quantified using primersand Taqman probe designed for GAPDH gene (Chromosome 12). Real time PCRis run with GAPDH and Amyloid (Chromosome 21) primers and probes beforerunning with Digital Array to confirm that the amplification regions areintact. To increase the signal from Chromosome 21, an additional set ofprimers and probes can be used.

One possible candidate is the following (See Blood 104(1):149-158(2004):

DSCR1 (Downs Syndrome Critical Region 1) Chr 21 SEQ ID NO:6: 5′ (probe)-AGG TTG TGA AAA CAG CAG CAA TGC AAT GT-(quencher) P3′ Forward: (SEQ IDNO:7) 5′CCA CAG GAA GCC GCC TAG T 3′ Reverse: (SEQ ID NO:8): 5′TGA GGGAAG AAA GGA AAC GCT 3′Amplification region (with primers underlined) SEQ ID NO: 9):CCACAGGAAGCCGCCTAGTGCAGAGAGGTTGTGAAAACAGCAGCAATGCAATGTGGAAATTGTAGCGTTTCCTTTCTTCCCTCA. An additional set of primers and probescan be designed to increase signal from the control (these primers canbe for chromosome 12 or any other chromosomes except Chromosome 21).

If an automated microfluidic device is used, appropriate channels andvalves are provided for introduction of PCR reactants and, if used, aprobe.

In the Examples below, a Fluidigm prototype DID chip was used. TheDigital Isolation and Detection (DID) chip works by partitioning asample/assay (TaqMan® assays) mixture into hundreds to tens of thousandsof reaction chambers, where real-time QPCR reactions are continuouslymonitored by a dynamic array reader. The DID chip described herecontains inputs for 12 sample/assay mixtures, and its architecturepartitions 7.5 μL of fluid for each input into 1,200 reaction chambers.These are shown as 12 panels in FIGS. 2 and 3. Instrumentation is usedto drive the sample/assay mixtures from the wells in the carrier intothe appropriate reaction chambers. As shown in FIGS. 2 and 3, whitespots indicate the location of reaction chambers positive for theindicated primer and dye. The sum of the positive wells in each sectionwill be consistent with the gene/chromosome copies that were measured inthe sample. The number of light spots shown represents the number ofpositive reaction chambers; no quantification was used in theseexperiments, and the results do not depend on quantization of a signalfrom an individual (discrete) sample mixture. Such quantization can beused, but can also be a source of error in methods that depend on this.

This chip is further described in Ottesen et al., “Microfluidic DigitalPCR Enables Multigene Analysis of Individual Environmental Bacteria,”Science 314:1464-1467 (Dec. 1, 2006). As discussed there, the DNA sampleis suspended in a PCR reaction buffer and loaded into the microfluidicdevice. The present work was done a more recent version of thatmicrofluidic device. This device is further described below. As analternative to the above protocol, one may use a kit with pre-optimizedreagents, such as the Qiagen QuantiTect Multiplex PCR Kit, whichcontains QuantiTect Multiplex PCR Buffer, having synthetic factor MP andan optimized combination of KCl and (NH₄)₂SO₄, which promote specificand stable annealing of primers to templates. This kit also containsHotStarTaq DNA Polymerase: Since this polymerase requires incubation at95° C. for activation, misprimed products and primer-dimers, which cancompete for reactants, are not formed during reaction setup.

One also uses the following anti-contamination procedures:

-   1. Use aerosol resistant pipette tips-   2. Preamplification treatment by use of uracil N-glycosylase, which    destroyed uracil-containing PCR products/RNA-   3. Negative water blank-   4. Negative blank gel slices-   5. Negative control panel on Digital Array

After extraction from blood and purification, the preferredconcentration of DNA sample should be ˜140-240 copies/μl, i.e., ˜70-120GE/μl. This corresponds to ˜3.4 to 2 μl of required template volume in adigital PCR reaction volume of 10 μl.

In this protocol and the following examples, the mixture of maternal andfetal genetic material obtained from the mother is diluted to achieve ahigh likelihood that only one target sequence will be present in a givensample to be analyzed. As shown in FIG. 1A, it is also possible to carryout this process with less dilution and less empty sample sites ifquantitation is used to distinguish a number of target sequences in asample.

IV. EXAMPLES

Presented below are data obtained from genomic DNA extracted from anormal human cell line and from a Down Syndrome cell line (trisomy 21).These cell lines were purchased from ATCC. Taqman PCR primers specificfor chromosome 21 and chromosome 12 were adapted from a reference:Zimmermann B et al, “Novel Real-Time Quantitative PCR Test for Trisomy21”. Clinical Chemistry. 48 (no. 2). 2002. 362-363. HEX(hexachloro-6-carboxyfluorescein) and FAM (6-carboxy-fluorescein) arewell known fluorescent dyes; BHQ® quencher is black hole quencher dye(BHQ, Biosearch Technologies, Novato, Calif.).

Amyloid Forward: 5′GGG AGC TGG TAC AGA AAT GAC TTC 3′ (SEQ ID NO:11)Amyloid Reverse: 5′TTG CTC ATT GCG CTG ACA A 3′ (SEQ ID NO:12) AmyloidProbe: 5′ (FAM) AGC CAT CCT TCC CGG GCC (SEQ ID NO:13) TAG G (BHQ)3′GAPDH Forward: 5′CCC CAC ACA CAT GCA CTT ACC 3′ (SEQ ID NO:14) GAPDHReverse: 5′CCT AGT CCC AGG GCT TTG ATT 3′ (SEQ ID NO:15)

GAPDH Probe: 5′ (HEX)AAA GAG CTA GGA AGG ACA GGC AAC TTG GC (BHQ)3′ (SEQID NO: 16) primers and probes were synthesized by IDT (Integrated DNATechnologies)). DNA samples were analyzed by digital PCR usingmicrofluidic Digital PCR on a Fluidigm® microfluidic chip having 12panels with 765 (wells) partitions each. Various mixtures of normal andDowns DNA (representing a mixture of fetal and maternal cells in a bloodsample) were analyzed. Small amounts of each template were pipetteddirectly into each PCR mix; alternatively, a mixture of templates couldbe prepared first, then pipetted into the PCR mix, which should yieldmore accurate results. The alternate method was used in theseexperiments. In these examples, trisomy is detected based on the numberof wells showing the triplicate chromosomal marker, i.e., the analysisillustrated in FIG. 1C. Intensity data of the triplicate chromosome arenot used, except as a ratio to a normal chromosome marker. Because thesample is dilute many of the wells will have no chromosome of interest(or marker fragment), as can be seen in FIGS. 2 and 3, which showphotographs of chips from Example 2.

Protocol: combine Primers 300 nM; Probes 150 nM; iTaq supermix with ROXor iQ supermix. Tween20 (0.1%); DNA template (2 μl, premixed with thedesired percentage of Downs DNA); Water (make up to total reactionvolume of 10 μl).

Each panel was loaded with reaction mix of 10 μl, and PCR was performedon a thermal cycler similar to the commercially available BIOMARK Systemfrom Fluidigm according to manufacturer's instructions. Cyclingconditions were: 98° C. 30 s, 97° C. 30 s, 95° C. 2 min, [56° C. 30 s,58° C. 30 s, 60° C. 30 s, 98° C. 15 s]×40 cycles, 60° C. for 10 min.

A MATLAB program was written to subtract the image of the chip takenbefore cycling from that taken at cycle 40 for each fluorescent channel.The number of positive wells in each fluorescent channel was counted.

Experiments were done with samples that contained 100% Downs, 60% Downs,50% Downs, 40% Downs, 30% Downs, and 0% Downs (i.e., 100% Normal) DNA.The results from these experiments are shown in FIG. 4, where each barand data point represents a different concentration of Down's DNA.

The data from the Data were analyzed as follows:

X*=number of HEX counts(Chromosome 12)

Y*=number of FAM counts(Chromosome 21)

There is a characteristic response for digital PCR. At low copy number,as described further in Warren L, Bryder D, Weissman I R, Quake S R.Transcription factor profiling in individual hematopoietic progenitorsby digital RT-PCR. See, PNAS 2006. 103: 17807-17812).

X=actual input copy number of Chromosome 12

Y=actual input copy number of Chromosome 21

X=log(1−X*/N)/log(1−1/N)

Y=log(1−Y*/N)/log(1−1/N)

N=total number of partition per panel=765

Confidence interval k=(Y−X)/sqrt(Y)

In FIG. 4, the confidence level obtained for a single chip was plottedagainst percent downs DNA and compared to the predicted confidenceinterval (line). The observed confidence interval at, for example 30%DNA, was less than predicted, but none the less showed a confidenceinterval of >1 for only one panel.

This calculation was done as follows:For 1 genomic equivalence,

Copy number of Chromosome 12=2(1−ε)+2ε=2

Copy number of Chromosome 21=2(1−ε)+3ε=2+ε

Difference between copy numbers of Chr21 and Chr12=ε

Where ε=fetal DNA/total free floating DNA*100%For m genomic equivalence

Y=copy number of Chromosome 21

X=copy number of Chromosome 12

Difference between copy numbers of Chr21 and Chr12=D=Y−X=mε

D=kσ _(y) =k*sqrt(Y), assuming that the distribution of Y follows thatof Poisson(mean standard deviation=Y)

k=mε/sqrt(Y)

m=Y/(2+ε)

k=sqrt(Y)*ε/(2+ε)

If ⅓ of the panel is used (i.e., 1 positive compartment in every 3compartments)

N=number of compartments

Y*=N/3

Y=log(1−Y*/3)/log(1−1/N)=log(⅔)/log(1−1/N)

k varies with N as shown in the graph.

The confidence interval k corresponds to the standard deviation, where ahigher standard deviation indicates a greater difference between thenormal and the Down's DNA. Even at the lowest concentration used (30%)and with only 10 panels analyzed, statistical analysis showed thefeasibility of the present method.

FIGS. 2 and 3 show results from 100% Downs samples, for easy of visualanalysis. In each panel, the number of white spots indicates thepositive wells for the markers tested. Chromosome 21 can be seen to havemore spots by simple visual observation, distinguishing the trisomicfrom the normal chromosome. In a 30% mixture (representing an enrichedmaternal blood sample), the results were analyzed statistically.

Table 1 below shows the results for each panel (numbered as in FIGS. 2and 3) in a single experiment using 30% Down's DNA.

TABLE 1 Panel Sample FAM HEX Ratio 1 Normal 221 213 1.04 2 Normal 254264 0.96 3 Normal 271 252 1.08 4 Normal 246 257 0.96 5 Normal 241 2381.01 8 30% Downs 270 222 1.22 9 30% Downs 219 194 1.13 10 30% Downs 249234 1.06 11 30% Downs 230 223 1.03 12 30% Downs 216 189 1.14

The “FAM” column shows the compartments (wells) positive for chromosome21, and the “HEX” column shows the compartments positive for chromosome12. The significance of the higher ratios in the Downs cases is shown inFIG. 4, and was also analyzed in a Student's T-test, with a value of0.036599344.

The above analysis shows that the statistical reliability of the presentmethod can be dramatically improved simply by increasing the number ofwells tested. Since about 240 genome equivalents is required per panel,and about 4,700 genome equivalents are found in a 20 ml sample, it ispossible, given the present description, to simply run additionalanalyses to increase statistical significance.

CONCLUSION

The present examples, methods, procedures, specific compounds andmolecules are meant to exemplify and illustrate the invention and shouldin no way be seen as limiting the scope of the invention, which isdefined by the literal and equivalent scope of the appended claims. Anypatents or publications mentioned in this specification are indicativeof levels of those skilled in the art to which the patent pertains andare intended to convey details of the invention which may not beexplicitly set out but would be understood by workers in the field. Suchpatents or publications are hereby incorporated by reference to the sameextent as if each was specifically and individually incorporated byreference and for the purpose of describing and enabling the method ormaterial referred to. The exemplary protocols given are for theconvenience of the reader and are not to be construed as necessary toone of ordinary skill in the art, given the teachings of the presentspecification regarding the various methods and materials to be used.

1. A method of differential detection of target sequences in a mixtureof maternal and fetal genetic material, comprising the steps of: a)obtaining maternal tissue containing both maternal and fetal geneticmaterial; b) distributing the genetic material into discrete samples,each sample containing on average not more than about one targetsequence per sample; c) measuring the presence of different targetsequences in the discrete samples; and d) analyzing a number of thediscrete samples sufficient to obtain results distinguishing differenttarget sequences.
 2. The method of claim 1 wherein one of the differenttarget sequences is diploid in maternal genetic material and aneuploidin fetal genetic material and another of the different target sequencesis diploid in both maternal and fetal genetic material.
 3. The method ofclaim 1 wherein the maternal tissue is maternal peripheral blood orblood plasma.
 4. The method of claim 1 wherein the discrete samples arein reaction samples selected from the group consisting of: wells in amicrotiter plate, aqueous phases in an emulsion, and reaction chambersin a microfluidic device.
 5. The method of claim 1 wherein the discretesamples are contacted with a plurality of PCR primers, including atleast one primer directed specifically to a maternal sequence and atleast one primer directed specifically to a fetal sequence, and furtherincluding the step of amplifying the maternal sequence and the fetalsequence.
 6. The method of claim 5 wherein said measuring the presenceof a target sequence further includes hybridizing it with a nucleic acidhaving a fluorescent label.
 7. The method of claim 6 wherein saidnucleic acid is in a sample for binding to a target sequence andgenerating a resulting change in fluorescence.
 8. The method of claim 1wherein the number of samples is at least about 10,000.
 9. The method ofclaim 1 wherein said measuring the presence of different targetsequences comprises the use of nucleic acids specific for humanchromosome 21 and specific for another human chromosome.
 10. The methodof claim 1 wherein said measuring the presence of different targetsequences comprises hybridization with nucleic acids which hybridize toa sequence selected from the group consisting of one or more of thegenes: CFTR, Factor VIII (F8 gene), beta globin, hemachromatosis, G6PD,neurofibromatosis, GAPDH, beta amyloid, and pyruvate kinase.
 11. Themethod of claim 1 wherein said measuring the presence of differenttarget sequences detects a sequence deleted in a human chromosomedeletion, is moved in a translocation or inversion, or is duplicated ina chromosome duplication, wherein said sequence is characterized in aknown genetic disorder in the fetal genetic material not present in thematernal genetic material.
 12. The method of claim 1 wherein saidmeasuring the presence of different target sequences detects a sequenceof a mutated form of a human gene listed in the OMIM Morbid map,http://www.ncbi.nlm.nih.gov/Omim/getmorbid.cgi.
 13. The method of claim1 further comprising the step of enriching the mixture for fetal geneticmaterial by size separation, whereby a preparation comprising only DNAfragments less than about 300 bp are used for measuring in step (c). 14.The method of claim 1 in which the measuring comprises direct sequencingof the genetic material.
 15. The method of claim 1 in which themeasuring comprises sequencing of amplified derivatives of the targetsequences clones or amplicons of the genetic material.
 16. The method ofclaim 16 where the amplified derivative is selected from the groupconsisting of an amplicon and a clone.
 17. The method of claim 1 inwhich the genetic material is cDNA derived from RNA in the tissue.
 18. Amethod of differential detection of target sequences in a mixture ofmaternal and fetal genetic material, comprising the steps of: a)obtaining from maternal plasma a mixture containing maternal and fetalgenetic material; b) distributing the mixture into at least five hundredseparate reaction samples, each reaction sample containing less thanabout one target sequence; c) hybridizing DNA in each separate reactionsample with nucleic acids hybridizing to one of two different targetsequences, one of which is used as a control sequence to detect targetsequally present on both maternal and fetal DNA and the other is used todetect a fetal genetic abnormality; and d) analyzing results of saidlabeling to obtain results distinguishing a difference in targetsequences in fetal DNA and maternal DNA.
 19. The method of claim 18wherein the distributing is into at least 10,000 separate reactionsamples.
 20. The method of claim 18 wherein said difference in targetsequences is a fetal aneuploidy.
 21. The method of claim 18 wherein saidprobes comprise a probe specific for human chromosome
 21. 22. A kit fordifferential detection of target sequences in maternal and fetal DNA ina mixed DNA sample, comprising: (a) primers specific for twochromosomes, one of which is possibly aneuploid and one of which ispresumed diploid; (b) a PCR reaction buffer for forming a PCR reactionsample with the primers; and (c) a size separation medium for separatingthe DNA sample into a fraction having less than about 1000 bp.
 23. Thekit of claim 22 where the primers comprise a pair of primers specific tochromosome
 21. 24. The kit of claim 22 further comprising a reactionchamber having at least 1,000 discrete reaction samples.